Uptake of hypoxanthine (Hx) and guanine (Gua) by isolated membrane vesicles of Salmonella typhimurium LT-2 is stimulated greater than 8-fold by PribosePP(PRPP). GMP and IMP, respectively, accumulate inside the vesicles. Initial rates of uptake into the vesicles are 100 pmoles/min/mg membrane protein and lead to greater than 12-fold concentration inside the vesicles. The Km for Hx is 10 microns M, the Km for guanine is 50 microns M, and the Km for PRPP when Hx is substrate is 90 microns M. Hx phosphoribosyltransferase (PRT) and GuaPRT associated with the vesicles is greater than 90% released by sonic treatment. 95% of HxPRT released, is recovered soluble after treatment while 43% of the GuaPRT released, is recovered soluble. Membrane vesicles of LT-2 ProAB47 exhibit less than 10% activity for gua uptake and gua PRT. When Pro AB47 vesicles are sonicated, however, both HPRT and GuaPRT appear in the soluble fraction. The recovery for HxPRT is similar to that observed in the wild-type (approximately 95%) while recovery of GuaPRT is 215%, or 5.0-fold greater ratio than found in the wild-type. Thus, the appearance of a substrate specificity (GuaPRT) only upon release of the enzyme from the membrane, suggests that the enzyme is constrained in situ in a more restricted conformation. Since cellular abilities parallel membrane specificity, we believe the purine PRTases are integral membrane components in situ and participate in group translocation.